Treatment of intracellular infection

ABSTRACT

An agent for combating an intracellular microbial infection comprises a phage component and, associated therewith, a targeting moiety which directs the agent to a target cell and initiates delivery of the phage into the target cell. Once inside the target cell, the phage causes lysis of a microorganism residing within the target cell. A mycobacteriophage is combined with a targeting moiety of transferrin. Compositions comprising the agent, methods of preparing said agent, and use of said agent for combating intracellular infections are also provided.

The present application is a 371 of PCT/GB00/01350 filed on Apr. 10,2000, and published in English on Oct. 19, 2000, the disclosure of whichis incorporated herein by reference in its entirety.

The present invention relates to an agent for causing lysis of amicroorganism residing within a cell, to a method for preparing saidagent, to compositions comprising said agent, and to the use of saidagent. In particular, the agent of the present invention is suitable forthe treatment of an intracellular infection by a microorganism.

Many microorganisms are capable of forming intracellular infections.These include: infections caused by species of Salmonella, Yersinia,Shigella, Campylobacter and Chlamydia. Live Salmonella and Yersinia cansurvive within the cells of mucosa of the gastrointestinal tract andfibroblasts, provide antigenic material continuously into the bloodcirculation and stimulate chronic inflammation and lead to arthritis;infections caused by the survival of Legionella pneumophila withinalveolar macrophages and epithelial cells; infections caused by thesurvival of Listeria monocytogenes within cell cytosol; infectionscaused by an intracellular protozoan Toxoplasma gondii; and infectionscaused by the intracellular survival of Bordetella species(macrophages), Staphylococcus aureus (epithelial cells) and Group Bstreptococci (macrophages). Some of these infections are exclusivelyintracellular, others contain both intracellular and extracellularcomponents. However, it is the intracellular survival cycle of bacterialinfection which is suspected as a main supportive factor for diseaseprogression.

Generally, these microorganisms do not circulate freely in the body, forexample, in the bloodstream. Accordingly, intracellular microorganismsare often not amenable to drug treatment regimes. Where drugs areavailable, this problem has been exacerbated by the development ofmultiple drug resistant. microorganisms. For similar reasons, vaccinetherapies are not effective against such intracellular microorganisms.Also, increased systemic concentration of antibiotics to improvebioavailability within cells may result in severe side effects.

As an example of an intracellular disease-causing microorganism,reference is made to Mycobacteria tuberculosis. This bacterium isresponsible for causing the disease tuberculosis which is responsiblefor more than three million deaths a year world-wide. M. tuberculosisinfects macrophage cells within the body. Soon after macrophageinfection, most M. tuberculosis bacteria enter, persist and replicatewithin cellular phagosome vesicles, where the bacteria are sequesteredfrom host defenses and extracellular factors.

A number of drug therapy regimes have been proposed for combating M.tuberculosis infections, with the best results to date having beenachieved with the drug isoniazid. As an alternative, bacteriophagetherapy has been suggested in the early 1980's based on results of thetreatment of experimental tuberculosis with rabbits infected with M.bovis BSG and M. microti, and guinea pigs infected with the humanpathogen M. tuberculosis strain H37Rv. However, the highest therapeuticeffect obtained with bacteriophage was not higher than that achievedwith isoniazid.

Phage, in particular bacteriophage, have been known for many years andhave been employed as delivery vehicles in conventional treatmentregiments for alleviating conditions associated with defective oraberrant cells.

For example, WO 98/05344 teaches the use of bacteriophage for deliveringan exogenous gene, such as a therapeutic polynucleotide, to a mammaliancell. Targeting of the bacteriophage to a pre-selected cell is achievedby use of a targeting moiety linked to the bacteriophage, said targetingmoiety effecting binding and initiating internalisation of thebacteriophage into the pre-selected cell. Once delivered to thepre-selected mammalian target cell, the exogenous genetic material canbe transcribed and translated, thereby increasing the concentration ofthe therapeutic molecule encoded by the therapeutic polynucleotide inthe target cell.

Aberrant cell treatment regiments such as those disclosed in WO 98/05344are conventionally known as gene therapy methods. Such regiments,however, do not address the problem and/or persistence of intracellularinfections by microorganisms.

WO 97/29185 teaches the preparation of recombinant phages, and the usethereof in the treatment or prophylaxis of bacterial infections.According to WO 97/29185, an anti-bacterial antibody is presented froman exposed surface of a bacteriophage, thereby rendering thebacteriophage capable of binding to and inhibiting growth of thetargeted bacterial cell. WO 97/29185 does not, however, teach how tocombat intracellular infections by microorganisms.

Additional background art relating to modified bacteriophage is providedin:

WO 99/10485, which teaches a bacteriophage system for identifyingligands susceptible to cell internalisation. Such ligands may providesuitable targets for bacteriophage gene delivery vehicles; and

WO 94/24959, which teaches a method of detecting compounds by utilisinga chemically modified lambdoid bacteriophage. In more detail, abacteriophage is modified to form a phage-target molecule complex, saidcomplex being non-infective. Upon challenge with a molecule of interest,the target molecule is cleaved and the bacteriophage becomes infective.Thus, the presence of a molecule of interest may be detected by thepresence of infective bacteriophage.

Neither of WO 99/10485 or WO 94/24959 addresses problems associated withmicrobial infections, least of all the problem of combatingintracellular microbial infections.

There is therefore a need for a system for combating intracellularinfections by microorganisms. In particular, there is a need for asystem for combating intracellular infections by mycobacteria.

The above problem is alleviated by the present invention which,according to a first aspect, provides an agent for causing lysis of amicroorganism residing within a target cell, comprising a targetingmoiety capable of binding to a target cell and a phage associated withthe targeting moiety, wherein following binding of the targeting moietyof the cell the phage enters the target cell and effects lysis of themicroorganism residing within the target cell.

The term “targeting moiety” means any structure which is capable ofbinding to the cell of interest. Examples include an antibody orfragment thereof, a receptor capable of binding to a ligand on the cellof interest, and a ligand capable of binding to a receptor on the cellof interest. Preferably, the targeting moiety is a ligand for acell-surface receptor. Good results have been achieved in a specificembodiment of the invention using a transferrin molecule as targetingmoiety. The targeting moiety need not demonstrate 100% specificity forthe cell of interest, though naturally a degree of specificity isdesirable for a highly efficient system. The targeting moiety may becapable of binding and internalisation, in which case the phage andtargeting moiety may be delivered as a complex (ie. associated) into thetarget cell. Identification of potential targeting moieties susceptibleto internalisation may be achieved by, for example, conventional methodssuch as those disclosed in WO 99/10485, or on a trial-and-error basis.Alternatively, the targeting moiety may be capable of binding but notinternalisation, in which case the phage alone may be delivered into thetarget cell.

The term “binding” includes any interaction between the targeting moietyand the cell of interest which permits the phage to be delivered intothe cell. This delivery process is one in which the whole phage entersthe cell of interest. The targeting moiety may become separated from thephage during this delivery process. Without being bound by any theory,it is believed that binding involves the formation of a complex betweenthe agent and a receptor present on the target cell. It is believed thatformation of the complex induces internalisation of the agent via areceptor-mediated delivery mechanism such as that utilised by nativeeukaryotic viruses (eg. adenovirus).

The term “associated” means any interaction between the targeting moietyand the phage such that the targeting moiety is capable of directing thephage to the cell of interest and when so directed the phage may bedelivered into the cell. Any one phage may be associated with one ormore targeting moieties. Where a given phage is associated with morethan one targeting moiety, each such moiety may bind to a differentcell-type. Alternatively, each targeting moiety preferably binds to thesame cell-type, although each may recognise a different site on the samecell-type.

The term “lysis” is used in this specification to include destruction ofthe microorganism through damage to or rupture of the microorganism cellwall. However, it is also intended to include any phage action whichcauses arrested growth and/or multiplication of the intracellularmicroorganism. In contrast to the phage of the present invention, whichare employed to combat intracellular microbial infections by effectinglysis of the microorganism in question, the prior art bacteriophagevectors employed in gene therapy regiments are non-lytic towardsmicroorganisms. Conventional gene therapy bacteriophage vectors arenon-lytic towards microorganisms to ensure that the natural bacterialflora of a mammalian host are unaffected by the bacteriophage duringgene transfer treatment regiments. In this respect, conventional genetherapy bacteriophage are often rendered abortive to lytic growth priorto use in gene therapy regiments. This may be achieved, for example, bymodifying bacteriophage tail proteins that are required for naturalphage transduction so that the bacteriophage is non-functional in aprokaryotic host, or by otherwise rendering the bacteriophage incapableof mediating injection of genetic material into a eukaryotic host cell.In contrast, the phage of the present invention are capable of naturalphage transduction and of effecting lysis of a microorganism.

Reference to phage throughout this specification includes recombinantphage and derivatives thereof which are capable of causing lysis of amicroorganism.

In operation of a specific embodiment of the invention, described belowin more detail, the targeting moiety is a ligand for a cell-surfacereceptor and is physically or chemically associated with a phage. Thisphage-targeting moiety combination is administered to cells infected byan intracellular microorganism and the phage enters the cells and lysesmicroorganism within those cells. In the case that the microorganism islocated within an intracellular compartment or vesicle the phage mayalso enter that internal compartment or vesicle. It is further preferredthat the targeting moiety binds to an internal compartment or vesicle inthe cell and within which the microorganism can reside. Thus, thetargeting moiety may be a ligand for a cell surface receptor and also aligand for a receptor on the surface of an internal compartment orvesicle. Following internalisation of the phage into a target cell, thepresence of a targeting moiety for a receptor on the surface of aninternal compartment or vesicle facilitates entry of the phage into theinternal compartment or vesicle where, once inside, it may exert a lyticeffect on the microorganism residing within the internal compartment orvesicle.

The targeting moiety for a cell surface receptor and the targetingmoiety for a receptor on the surface of an internal compartment orvesicle may be the same or different. In the latter embodiment, theagents of the present invention may be modified such that the targetingmoiety for the receptor on the internal compartment or vesicle isfunctional only following entry of the phage into a target cell. Thismay be achieved, for example, by employing a cell-specific promoter toensure that the targeting moiety for the receptor on the surface of theinternal compartment or vesicle is expressed only after the phage hasbeen delivered to that specific cell-type. Alternatively, the targetingmoiety for the receptor on the surface of the internal compartment orvesicle may be obscured (eg. by steric hindrance) on the surface of thephage prior to use thereof in accordance with the present invention.However, the targeting moiety for the receptor on the surface of theinternal compartment or vesicle may be made accessible (eg. by cleavageor other modification of the targeting moiety for the cell surfacereceptor) during the internalisation process following binding of theagent to the target cell.

In one embodiment of the present invention, once the phage of thepresent invention has been delivered to the target cell of interest itremains as a separate entity and does not (or any part thereof)integrate into the target cell's genome.

In another embodiment, the phage contains substantially no exogenous(ie. non-phage) nucleic acid. In a further embodiment the phage containssubstantially no exogenous nucleic acid other than that coding for thetargeting moiety.

In one embodiment, the phage comprises substantially no exogenoustherapeutic polynucleotides capable of mediating a therapeutic benefitin a recipient of the polynucleotide or product thereof. A “therapeuticpolynucleotide product” refers to a molecule produced as a result oftranscription or translation of the therapeutic polynucleotide.Therapeutic polynucleotide products include transcription products (eg.antisense mRNA and catalytic RNA), and translation products (eg.proteins or peptides) of the therapeutic polynucleotide.

The targeting moiety may be chemically linked to the phage. This may beachieved, for example, via a linker molecule (eg. a short peptide) or byother covalent means, for example, via a disulphide bridge. Thetargeting moiety may be bound to any part of the phage. The fusionbetween a targeting moiety and a phage should impair neither the bindingof the targeting moiety to the cell membrane receptor nor the binding ofthe phage to its bacterial receptor. In relation to the targetingmoiety, the chemical linkage procedure should not significantly alterthe binding site to the cell receptor, e.g. by altering its conformationor by imposing structural rigidity. The transferrin chemical linkageillustrated in the present application does not prevent its binding toits receptor. The selected targeting moiety preferably demonstratesconformational stability. The chemical linkage construct preferablyplaces the targeting moiety at a distance from the phage body sufficientto provide the targeting moiety with a degree of rotational flexibilityso as to preserve maximum interaction of the targeting moiety with itsreceptor. The preferred means for chemical linkage is heterofunctionalcrosslinking via a disulfide bridge.

Alternatively, the targeting moiety may be physically mixed with thephage. In this case, the target moiety may become physically adsorbed tothe phage by, for example, hydrogen-bonding, hydrophobic/hydrophilicbonding, van der Waal's forces, electrostatic forces or other chargedassociations. For example, the phage may carry a negative surface chargeand the targeting moiety would then preferably carry a positive surfacecharge. Alternatively, at the selected pH (eg. pH 7-8, preferablyapproximately pH 7.5) the phage carries a positive charge sufficient tobind to a more negatively charged targeting moiety.

There are several ways of chemical linking of a targeting moiety to thephage via a disulfide bridge, examples of which are given below. Whilsttransferrin (Tf) is illustrated below as the targeting moiety, the samelinking means are equally applicable to other types of targeting moiety.

One embodiment is first to introduce free sulfhydril groups (—SH) in thetransferrin molecule which does not have such groups in its nativestate. This can be done, eg. by thiolation of one or more free aminogroups of the transferrin molecule with a reagent (eg. 2-iminothiolane)which modifies these groups and introduces —SH groups at these positions(this leads to Tf—SH). By changing the ratio of transferrin to2-iminothiolane, temperature and pH of the reaction, modification can beachieved that will not impair transferrin binding to its receptor.Preferably 5-10 SH groups per Tf molecule are so modified. Then modifiedtransferrin is purified from the low molecule weight reagent by gelfiltration, eg. with Sephadex G-50 column and concentrated to about 1-3mg/ml over a microfiltration membrane with a 30 kDa cut-off.

The phage is pre-treated by mixing with a hetero-bifunctionalcross-linking agent (eg. Sulfo-SMCC, sulphosuccinilimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate) which covalently bindsto free-NH₂ groups on the phage, leaving in the solution phage havingactive maleimide groups which are able to react with a —SH group fromthe thiolated transferrin. The degree of phage modification may becontrolled by the ratio of the crosslinking agent to phage. Thisreaction should take place preferably at a temperature in the interval4-15° C. to preserve phage stability and to provide a good rate ofmodification. At the end of the reaction, modified phage is isolatedfrom the reaction mixture by gel filtration on Sepharose 6B column andconcentrated by membrane filtration.

At the next step, Tf—SH is mixed with an activated phage and the product(Tf-S-S-linker-Phage) is again isolated from Tf—SH by gel filtration inthe same column. The presence of non-reacted phage can be afforded sinceit can only enhance the biological activity of the final preparation.

Another strategy of developing Tf-S-S-linker-Phage product can involvethe activation of Tf free —NH2 groups and cross-linking activatedtransferrin with —SH groups at the phage surface. The outcome of thisprocess depends on the availability and accessibility of —SH groups atthe phage surface.

The formation of Tf-phage physical aggregate depends mainly on theirelectrostatic charge at the pH of the mixture (this is' preferably takenas a physiological pH).

The present invention has application in the treatment of anymicroorganism within a cell. In use of the invention, a phage capable oflysing the microorganism is identified and associated with a targetingmoiety capable of directing the phage to the infected cell, preferablyto a specific compartment of the infected cell which contains aninfectious agent. Thus, the agent of the present invention may beemployed to treat an intracellular infection by a virus or by abacterium. In one embodiment, this may be achieved by selection of atargeting moiety which is the same as the receptor employed by theinfectious agent of interest. The agent of the present invention wouldthen follow the same intracellular route as the infectious agent. By wayof example, complement receptors CR1 and CR3 are known as a gate for M.tuberculosis infection and therefore complement components such as C2aand C3B are targeting moiety candidates for phage modification. Anothercandidate which can be internalised via CR3 is Bordetella pertussishaemaglutinin. Another group of targeting moieties comprises those usedby infectious agents during their intracellular persistence and/or whichare required for their replication. Transferrin is one example of such amoiety. This protein is required for providing M. tuberculosis with ironwhich is critical for the bacterium's intracellular survival.

The present invention is suitable for treating a number of intracellularinfections. These include infections caused by Salmonella, Yersinia,Shigella, Campylobacter and Chlamidia. Live Salmonella and Yersinia cansurvive within the cells of mucosa of the gastrointestinal tract andfibroblasts, provide antigenic material continuously into the bloodcirculation and stimulate chronic inflammation and lead to arthritis.Also infections caused by the survival of Legionella pneumophila withinalveolar macrophages and epithelial infections caused by the survival ofListeria monocytogenes within cell cytosis infections caused by anintracellular protozoan Toxoplasma gondii, and infections caused by theintracellular survival of Bordetella species (macrophages),Staphylococcus aureus (epithelial cells) and Group B streptococci(macrophages). Some of these infections are exclusively intracellular,others contain both intracellular and extracellular components. However,it is the intracellular survival cycle of bacterial infection which issuspected as a main supportive factor for disease progression.

The present invention is also applicable for suppression ofintracellular persistence, for example, within macrophages of otherbacteria such as Leishmania donovani, Legionella pneumophila, Bordetellapertussis and other species of bordetellae, Group B streptococci,Salmonella species, Chlamydia and Borrelia burgdorferi. This can beachieved with the use of the macrophage-specific delivery moiety and alytic bacteriophage specific for the microorganism.

The present invention maybe used against intracellular viruses. Theremaybe an advantage in using a targeting moiety linked eg. to an antibodyto a specific viral protein or to a short anti-sense DNA fragment. Suchfusion construction may be delivered into a target cell as describedpreviously.

Where the microorganism is a bacterium, the phage for use in the agentof the present invention is a bacteriophage. Bacteriophages are phageswhich parasitise bacteria. They typically comprise a head containinggenetic material (usually DNA; though occasionally RNA), enclosed by awall of protein which is usually prolonged into a hollow tail. Abacteriophage initiates infection by attaching itself by its tail to thewall of a bacterial cell. Through enzyme action, the wall is perforatedand bacteriophage genetic material passes through and into the bacterialcell. The bacteriophage genetic material then organises the bacterium tomake more bacteriophage genetic material which assembles withbacteriophage head and tail to form assembled particles. These assembledparticles are then released by lysis of the host bacterial cell.Bacteriophage are typically highly specific, with each kind ofbacteriophage typically infecting only one bacterial species or strain.

In one embodiment, the bacteriophage is preferably a lyticbacteriophage.

Preferably the bacteriophage is a mycobacteriophage. Themycobacteriophage is preferably specific for a particular species ofmycobacteria. Most preferably the mycobacteriophage is selected from thegroup consisting of lytic mycobacteriophages L29, D34, DS-6A.

During infection by mycobacteria the bacteria enter macrophages viareceptor-mediated phagocytosis which may involve severalmycobacteria-specific receptors on the macrophage membrane. Followinginitial interaction with receptor(s), the mycobacteria enter the earlyphagosome, arrest its maturation and sequester it from the terminalphagocytic organelles e.g. lysosomes. This prevents fusion of theinfected phagosome with lysosome and subsequent lysosome-directed lysisof the mycobacteria. It is by this mechanism that mycobacteria form anintracellular infection within a vesicle of the macrophage and avoid thehost cell's immune system.

Mycobacteria infect monocytes and macrophages. Thus, when selecting atargeting moiety for use in an agent for treating a mycobacterial cellinfection, that targeting moiety should bind to a monocyte and/or amacrophage.

Suitable targeting moieties include Bordetella pertussis filamentoushaemagglutinin which binds to complement receptor CR3 and can beinternalised via a receptor-medicated endocytosis mechanism; complementcomponent C3; antibody to C3 which can form an agent capable of bindingto C3 in human sera and directing phage internalisation through the CR3receptor; and ligands to macrophage receptors specific to mycobacteria(eg. mannose receptor, surfactant protein receptor, CD14 etc) which canbind receptors and be internalised. A transferrin molecule or a partthereof or a mutant or derivative thereof is a preferred targetingmoiety. For sequence details of transferrin, reference is made to Uzan,G., Frain, M., Park, I., Besmond, C., Maessen, G., Trepat, G. S., Zakin,M. M., and Kahn, A. (1984) Molecular cloning and sequence analysis ofcDNA for human transferrin Biochem Biophys. Res. Commun. 119, 1;273-281; and Welch, S. (1990) A comparison of the structure andproperties of serum transferrin from 17 animal species Comp. Biochem.Physiol.-B 97(3); 417-27.

According to a second aspect of the present invention, there is provideda method for preparing the agent according to the above definitions,comprising contacting the targeting moiety and the phage such that thetargeting moiety becomes associated with the phage.

A large stock of phage may be readily produced. Reference is now made tomycobacteriophage production, although similar scale-up procedures wouldbe equally applicable for other phages.

Phage stock may be produced by the infection of a liquid culture of M.tuberculosis or another auxiliary mycobacterium strain, removing celldebris by centrifugation, phage concentration (eg. by membranefiltration, PEG precipitation, centrifugation etc.,) followed by phagepurification (eg. by gel filtration, additional membrane filtrationetc.) from components (eg. proteins, polypeptides, salts, etc.) whichcan interfere with its chemical modification. This process can be easilyscaled up by using filtration and chromatographic devices with requiredthroughput.

The stock of phage may then be mixed with the targeting moiety ofinterest.

Transferrin is now illustrated simply as an example of a targetingmoiety. The bonding by physical adsorption requires only a mixing ofphage and targeting moiety which have sufficiently different isoelectricpoints (eg. transferrin, which molecule has pl=5.5, and a phage with plabove 7). The Tf-Phage complex appears stable under physiologicalconditions and it may be easily separated from the free Tf. The presenceof free phage can be afforded since it will only enhance the biologicalactivity of the preparation.

According to a third aspect of the present invention, the agent isprepared by bonding a targeting moiety to a phage. Examples of thepreferred bonding procedure have been discussed above.

According to a fourth aspect of the present invention, there is provideda chimeric phage capable of causing lysis of a microorganism within acell, comprising a targeting moiety capable of binding to the cell aspart of a tail or coat protein of the phage.

According to a fifth aspect of the present invention there is provided amethod for preparing the chimeric phage described above.Mycobacteriophages are illustrated as an example of this aspect of thepresent invention. Using mycobacteriophages which genomes have beenfully sequenced and characterised (eg. phage L5 or d29), primers can begenerated to identify DNA domains which encode “functionally silent”surface membrane proteins within the phage of interest. These can beexcised and replaced with a DNA domain encoding the receptor bindingmoiety (eg. binding domain of transferrin). Accordingly, novel“designed” chimeric phages may be produced which combine the replicationfunction and the receptor binding function. Phages can be accumulated asdiscussed above and used as therapeutic agents against intracellularpathogens.

According to a sixth aspect of the present invention there is provided acomposition comprising an agent of the present invention or a chimericphage of the present invention, and a pharmaceutically acceptablecarrier. The composition is preferably a liquid mixture of agent orchimeric phage and stabiliser which will stabilise the phage duringstorage in a nebuliser. The mixture may also contain another agent thatwill prevent phage aggregation.

Many intracellular infections caused by microorganisms are contracted byinhalation Many such intracellular infections are therefore concentratedin and around lung tissues. In particular, tuberculosis (caused by M.tuberculosis) is contracted in this manner. According to preferredembodiment, the composition according to the present invention isprovided in an aerosol form.

Reference is now made to the following examples.

Example 1 Modified Lytic Anti-M. tuberculosis Phage as an Agent for theTreatment of Tuberculosis.

This example describes the preparation of a lytic anti-M. tuberculosisphage (phage D34) that was modified either by absorption or chemicallyto carry a surface polypeptide that provides phage binding,internalisation and delivery into the phagosome within infectedmonocytes/macrophages. Once within the phagosome, the phage displays itslytic characteristics and either damages or lyses intraphagosomalmycobacteria.

When human monocyte/macrophage cells U937 infected with M. phlei weretreated with a sample of modified phage (columns 3 and 4 in Table 1),the number of alive intracellular mycobacteria plated from macrophagelysates was less as compared to that from lysates without phagetreatment or treatment with a non-modified phage (columns 5 and 2 inTable 1). The strongest effect was achieved when the targeting moiety(transferrin) was added to,a phage by physical absorption. As the resultof a physical modification, phage was two-fold more effective ascompared to non-modified phage. The latter level is known to be at leastequal to the best result achieved with antibiotics, eg. isoniazid.

These in vitro data demonstrate that phage modification with a specificdelivery targeting moiety enhance phage capability to reduce bacterialload within infected macrophages. The phage therapy with modified phagecan be efficient against M. tuberculosis infection and will help theimmune system to neutralise and control the infection and, eventually,tuberculosis.

TABLE 1 The Effect of Transferrin (Trf)-modified and non-modified phageon U937 infection with Mycobacterium phlei. Phage, Phage, Phage, Phage−Trf +nMTrf +Trf No Phage Titre 8.4 × 10⁶ 4.4 × 10³ 4.3 × 10⁴ — (1/ml)Viable cells 422 222 377 638 (1/ml) Viable cells/ 0.66 0.34 0.59 1.0Viable cells (−Phage) Differentiated U937 cells were used (4.8 × 10⁵cells/ml, 98% alive)

The above M. phlei-infected monocyte/macrophages in example 1 weresuspended for 1 hour in RPMI media containing gentamycin 4 mg/ml, washed3 times with PBS and resuspended in RPMI prior to treatment with themodified phage. This was to ensure that surface bound or released M.phlei bacteria (ie. non-intracellular mycobacteria) did not mask theresult of the example.

What is claimed is:
 1. A mycobacteriophage that includes a ligandassociated with the mycobacteriophage, wherein the ligand is atransferrin molecule, or a fragment thereof that binds to a transferrinreceptor on a macrophage or monocyte, and wherein following binding ofthe ligand to the macrophage or monocyte the mycobacteriophage entersthe macrophage or monocyte and effects lysis of a microorganism residingwithin the macrophage or monocyte.
 2. The mycobacteriophage of claim 1,wherein the microorganism is a bacterium.
 3. The mycobacteriophage ofclaim 2, wherein the bacterium is Mycobacterium tuberculosis.
 4. Themycobacteriophage of claim 1, wherein the mycobacteriophage is selectedfrom the group consisting of L29, D34 and DS-6A.
 5. A method ofpreparing the mycobacteriophage of claim 1, comprising contacting aligand, which is a transferrin molecule or a fragment thereof that bindsto a transferin receptor on a macrophage or monocyte, with amycobacteriophage such that the ligand becomes associated with themycobacteriophage.
 6. A method of preparing the mycobacteriophage ofclaim 1, comprising bonding a ligand, which is a transferrin molecule ora fragment thereof that binds to a transferin receptor on a macrophageor monocyte, to a mycobacteriophage.
 7. A composition comprising themycobacteriophage of claim 1, and a pharmaceutically acceptable carrier.8. The composition of claim 7 for aerosol delivery.
 9. A method oftreating an intracellular infection by a microorganism, comprisingadministering to a patient an effective amount of a mycobacteriophageaccording to claim 1, wherein the mycobacteriophage enters themacrophage or monocyte and effects lysis of the microorganism residingwithin the macrophage or monocyte.
 10. A mycobacteriophage that includesa ligand associated with the mycobacteriophage, wherein the ligand is atransferrin molecule or a fragment thereof that binds to a transferrinreceptor on the surface of a macrophage or monocyte said ligand beingexogenous with respect to the mycobacteriophage, and wherein followingbinding of the ligand to the macrophage or monocyte themycobacteriophage enters the macrophage or monocyte and effects lysis ofa microorganism residing within the macrophage or monocyte.